The human parvovirus adeno-associated virus (AAV) is non-pathogenic and integrates into a defined region of chromosome 19 to establish latency. The ability of AAV to integrate in a site-specific manner into the host genome is unique and has not been demonstrated for any other virus. AAV therefore provides an interesting model to study DNA integration and a potentially useful vector system for gene therapy. The region of chromosome 19 where AAV integrates has been named AAVS1. AAV integration into AAVS1 results in extensive rearrangements of the integration target sequence. Using an episome-based assay it was found that integration of wild-type AAV is directed by AAVS1 sequences. Further studies with this assay showed that the minimal recombination signals sufficient to mediate site-specific integration consist of (i) a Rep binding site (RBS) that is recognized by the viral Rep protein, and (ii) a terminal resolution site (TRS) where Rep introduces a site- specific nick. However, the episome-based assay is limited as a model to study the requirements for AAV DNA integration into the human genome, because of the inherent episomal replication and transcription. Furthermore, this assay is not suited to address the rearrangements associated with integration because of the requirements of the episomal shuttle vector to be propagated in E. coli. The objectives of this proposal are: (i) to define the requirements for site-specific integration of AAV into the human genome, (ii) to characterize the rearrangements resulting from AAV integration and (iii) to determine transcriptional activity of a transgene at different chromosomal loci. The overall goal is to establish feasibility of re-targeting AAV integration into selected chromosomal sites of therapeutic value. To determine whether RBS and TRS are sufficient to mediate integration within any chromosomal locus, we propose to establish clonal cell lines of human embryonic kidney cells containing AAVS sequences at sites other than chromosome 19 (AAVSN). Individual clones will be tested by Southern blot analysis to identify AAVSN sites not linked to the parental site. AAVSN sites will be mapped by fluorescent in situ hybridization (FISH). AAV integration into AAVSN loci will be studied using recombinant AAV containing the genes for Rep and neomycin resistance. The viral-cellular junctions at AAVSN sites will be isolated and the extent of chromosomal rearrangements will be assessed by sequence analysis. Transcriptional activity of a marker gene will be determined for different AAVSN loci and compared to the parental AAVS1.